Project description:Deciphering the genetic basis of plant secondary metabolism will provide useful insights for genetic improvement and enhance our fundamental understanding of plant biological processes. Although citrus plants are among the most important fruit crops worldwide, the genetic basis of secondary metabolism in these plants is largely unknown. Here, we use a high-density linkage map to dissect large-scale flavonoid metabolic traits measured in different tissues (young leaf, old leaf, mature pericarp, and mature pulp) of an F1 pseudo-testcross citrus population. We detected 80 flavonoids in this population and identified 138 quantitative trait loci (QTLs) for 57 flavonoids in these four tissues. Based on transcriptional profiling and functional annotation, twenty-one candidate genes were identified, and one gene encoding flavanone 3-hydroxylase (F3H) was functionally verified to result in naturally occurring variation in dihydrokaempferol content through genetic variations in its promoter and coding regions. The abundant data resources collected for diverse citrus germplasms here lay the foundation for complete characterization of the citrus flavonoid biosynthetic pathway and will thereby promote efficient utilization of metabolites in citrus quality improvement.
Project description:Citrus and most other fruit crops are commercially propagated via grafting, which ensures trees have consistent fruit traits combined with favorable traits from the rootstock such as soil adaptability, vigor, and resistance to soil pathogens. Breeding new rootstocks requires careful agronomic evaluations, and widespread use of new rootstocks and scions requires graft compatibility with commercially important scions and rootstocks. Graft incompatibility can occur when the scion and rootstock are not able to form a permanent, healthy union. Understanding and preventing graft incompatibility is therefore of paramount importance in the breeding of new fruit cultivars and in the choice of scion and rootstock by growers. The rootstock US-1283 is a citrandarin generated from a cross of ‘Ninkat’ mandarin (Citrus reticulata) and ‘Gotha Road’ #6 trifoliate orange (Poncirus trifoliata). It was released in 2014 after years of field evaluation because of its superior productivity and good fruit quality on ‘Hamlin’ sweet orange (C. sinensis) under Florida’s growing conditions. Subsequently, it was observed that trees of ‘Bearss’ lemon (C. limon) and ‘Valencia’ sweet orange (C. sinensis) grafted onto US-1283 exhibited apparent incompatible and unhealthy growth near the graft union. The incompatibility manifested as stem grooving and necrosis underneath the bark on the rootstock side of the graft. A genetically similar citrandarin rootstock, US-812 (C. reticulata ‘Sunki’ × P. trifoliata ‘Benecke’), is fully graft compatible with the same scions. Transcriptome analysis was performed on the vascular tissues above and below the graft union of compatible US-812 and incompatible US-1283 graft combinations with ‘Bearss’ and ‘Valencia’ to identify expression networks associated with incompatibility and help understand the processes and potential causes of incompatibility in citrus. Transcriptional reprogramming was stronger in the incompatible rootstock than in the grafted scions. Functional analysis of the transcriptional events below the graft unions of US-1283 incompatible combinations revealed differentially expression genes (DEGs) associated with oxidative stress and plant defense, among other pathways, similar to a pathogen-induced immune response localized to the rootstock, although no known pathogens were detected in the assayed plants. These changes were not observed above the graft unions.Differentially expressed genes (DEGs) in US-1283, but not the scions, were associated with oxidative stress and plant defense, among others, similar to a pathogen-induced immune response localized to the rootstock. No pathogen infection was detected. It is hypothesized this response could have been triggered by signaling miscommunications between rootstock and scion either through 1) unknown molecules from the scion that were perceived as danger signals by the rootstock, 2) missing signals from the scion or missing receptors in the rootstock necessary for the formation of a healthy graft union, 3) the overall perception of the scion by the rootstock as non-self, or 4) a combination of the above.
Project description:Huanglongbing (HLB), caused mainly by 'Candidatus Liberibacter asiaticus' (CLas), is the most devastating citrus disease because all commercial species are susceptible. HLB tolerance has been observed in Poncirus trifoliata and their hybrids. A wide-ranging transcriptomic analysis using contrasting genotypes regarding HLB severity was performed to identify the genetic mechanism associated with tolerance to HLB. The genotypes included Citrus sinensis, Citrus sunki, Poncirus trifoliata and three distinct groups of hybrids obtained from crosses between C. sunki and P. trifoliata. According to bacterial titer and symptomatology studies, the hybrids were clustered as susceptible, tolerant and resistant to HLB. In P. trifoliata and resistant hybrids, genes related to specific pathways were differentially expressed, in contrast to C. sinensis, C. sunki and susceptible hybrids, where several pathways were reprogrammed in response to CLas. Notably, a genetic tolerance mechanism was associated with the downregulation of gibberellin (GA) synthesis and the induction of cell wall strengthening. These defense mechanisms were triggered by a class of receptor-related genes and the induction of WRKY transcription factors. These results led us to build a hypothetical model to understand the genetic mechanisms involved in HLB tolerance that can be used as target guidance to develop citrus varieties or rootstocks with potential resistance to HLB.
Project description:Morphological observation of arbuscular mycorrhizal fungi (AMF) species in rhizospheric soil could not accurately reflect the actual AMF colonizing status in roots, while molecular identification of indigenous AMF colonizing citrus rootstocks at present was rare in China. In our study, community of AMF colonizing trifoliate orange (Poncirus trifoliata L. Raf.) and red tangerine (Citrus reticulata Blanco) were analyzed based on small subunit of ribosomal DNA genes. Morphological observation showed that arbuscular mycorrhizal (AM) colonization, spore density, and hyphal length did not differ significantly between two rootstocks. Phylogenetic analysis showed that 173 screened AMF sequences clustered in at least 10 discrete groups (GLO1~GLO10), all belonging to the genus of Glomus Sensu Lato. Among them, GLO1 clade (clustering with uncultured Glomus) accounting for 54.43% clones was the most common in trifoliate orange roots, while GLO6 clade (clustering with Glomus intraradices) accounting for 35.00% clones was the most common in red tangerine roots. Although, Shannon-Wiener indices exhibited no notable differences between both rootstocks, relative proportions of observed clades analysis revealed that composition of AMF communities colonizing two rootstocks varied severely. The results indicated that native AMF species in citrus rhizosphere had diverse colonization potential between two different rootstocks in the present orchards.
Project description:MicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation. miRNAs have been shown to control many genes involved in various biological and metabolic processes. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants. In this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata) an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues, Based on sequence similarity and hairpin structure prediction, we found that 178,102 reads representing 89 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates, whose precursors were all potentially generated from citrus ESTs. And of them five miRNA* sequences were also sequenced. These sequences had not been described in other plant species and accumulation of these 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes included one encoding IRX12 copper ion binding/ oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata.
Project description:MicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation. miRNAs have been shown to control many genes involved in various biological and metabolic processes. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants. In this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata) an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues, Based on sequence similarity and hairpin structure prediction, we found that 178,102 reads representing 89 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates, whose precursors were all potentially generated from citrus ESTs. And of them five miRNA* sequences were also sequenced. These sequences had not been described in other plant species and accumulation of these 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes included one encoding IRX12 copper ion binding/ oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.
Project description:A microarray containing 62,876 unigenes selected from CitEST database and prepared by Nimblegen Systems was used for identifying candidate resistance genes against P. parasitica at 48 hours after inoculation Four resistant and four susceptible F1 hybrids were selected from the population derived from the cross between Citrus sunki Hort. ex. Tan. and Poncirus trifoliate (L.) Raf cv. Rubidoux, respectively susceptible and resistant to P. parasitica. It was proposed that differentially expressed genes between resistant and susceptible hybrids and their parents provide essential candidates for identifying transcripts involved in disease resistance